A wide variety of tests have been developed for the determination of pregnancy. These methods, in general, involve the testing of blood or urine for levels of pregnancy antigens or other compounds which are indicative of pregnancy. Ectopic pregnancies, however, are not reliably distinguished from normal pregnancies by these methods, and ectopic pregnancies remain a major cause of morbidity and mortality for women.
Commercial early pregnancy determinations include the rabbit ovulation test of urine (5.5 wk), rat ovarian hyperemia test of urine (5.5 wk), hemagglutination inhibition latex particle test of urine (5.5 wk), RIA (radioimmunoassay) test of blood using competition with .sup.125 I labeled hCG for anti-(hCG) antibodies (3.5 wk), RIA test of blood using competition with .sup.125 I labeled hCG for anti-(.beta.-hCG) antibodies (3.5 wk), and RRA (radioreceptor assay) test of blood using competition with .sup.125 I labeled hCG for specific receptor sites. The RIA tests have been refined to provide qualitative results the same day but require an additional day to determine the exact titer of hCG or .beta.-hCG in the serum. The RRA assay is more rapid, but is currently less sensitive than the RIA test.
Home pregnancy tests for hCG in urine include a variety of enzyme immunoassays, hemagglutination inhibition, and antibody-antigen agglutination tests which are effective to indicate pregnancy from 0 to 7 days after a missed period. Confirmation by physician is recommended, particularly to determine abnormal gestation such as ectopic pregnancy.
Previously reported methods for determining ectopic pregnancies are summarized by Barnea, E. et al in ECTOPIC PREGNANCY. DeCherney, A. (editor) Rockville: Aspen Publications pp 65-80 (1986) and Seppala, M. et al in Clin.Obstet.Gyn. 30:148-154 (1987), and the reference publications listed in both of these publications. These methods require a comparison of the level of pregnancy antigens such as hCG or SP1 in blood or urine with normal pregnancy levels. Since the rise in level of pregnancy antigens varies from patient to patient and is a function of the time from conception, even the most precise of these methods is incapable of indicating ectopic pregnancy with an adequate level of confidence. RIA (radioimmunoassay) test of blood using competition with .sup.125 I labeled hCG for anti-(hCG) antibodies, RIA test of blood using competition with .sup.125 I labeled hCG for anti-(beta-hCG) antibodies, RRA (radioreceptor assay) test of blood using competition with .sup.125 I labeled hCG for specific receptor sites, and a variety of EIA (enzyme immunoassay) methods have been developed for accurately determining levels of hCG and SP1 in the blood. The RIA tests have been refined to provide qualitative results the same day but require an additional day to determine the exact titer of hCG or beta-hCG in the serum. The RRA assay is more rapid, but is currently less sensitive than the RIA test. These tests are relevant to the detection of ectopic pregnancies because the hCG values are, in general, lower in ectopic pregnancies than in normal gestation when these values are adjusted for gestational age. Theoretically, detection of abnormally low hCG levels provide an indication of possible ectopic pregnancy. When combined with ultrasound and laparoscopy, ectopic pregnancies can be verified with greater reliability.
Barnea, E. et al, J.Clin.Endocrinol.Metab. 62:529-531 (1986) reports that plasma estradiol, progesterone, free alpha-human chorionic gonadrotropin (alpha hCG), and hCG were measured in normal and confirmed tubal pregnancy patients, and free alpha hCG and hCG level increases were higher with ectopic pregnancy than with normal pregnancy. Plasma estradiol and progesterone levels were lower in patients with ectopic pregnancies. Olson, C. et al, J. Repro.Med. 28:838-842 (1983) reports that plasma beta-hCG levels are lower with ectopic pregnancies but was not effective to detect all ectopic pregnancies. A double antibody immunoassay test (SERONO BETA-III) was used. Okamoto, S. et al, Brit.Med.J. 295:667-670 (1987) reports that plasma .beta.-hCG testing could be effective for identifying ectopic pregnancies, but that in order to achieve a sensitivity of 100%, the positive threshold level must be so low that the specificity level falls to 68 percent with increased false positives. They used a three hour radioimmunoassay method. U.S. Pat. Nos. 4,016,250 and 4,094,963 describe the testing of hCG, LH (luteinizing hormone), PRL (prolactin) and hCG-like materials in aqueous samples (blood, serum and urine) using corpus luteum receptor sites for these materials in an RRA procedure. Results of studies with tubal ectopic pregnancies using this procedure are reported.
Fletcher, J. Primary Care. 13:667-677 (1986) reviews the bioassays and immunoassays which have been developed for pregnancy detection. The commercially available radioimmunoassays and enzyme-linked immunoassays (EIA or ELISA) and the advent of home pregnancy testing kits are described. Although increased detection of ectopic pregnancies was reported measuring hCG levels in urine, citing a study by Buck, R. et al, Clin.Chem. 32:879 (1986), Fletcher notes that low hCG values do not distinguish between ectopic pregnancy and impending abortion.
Basil Ho Yuen in Obst.Gyn.Fertil. 8:22-25 (1985) summarizes the findings of Braunstein, G. et al "First trimester chorionic gonadotropin measurement as an aid in the diagnosis of early pregnancy disorder." Am.J.Obstet.Gynecol. 131:25 (1978), and Braunstein, G. et al "Predictive value analysis of measurement of human chorionic gonadrotropin, pregnancy specific .beta..sub.1 -glycoprotein, placental lactogen, and cystine aminopeptidase for the diagnosis of ectopic pregnancy." Fertil.Steril. 39:62 (1983). A specificity of only 62.5 percent was reported measuring .beta.-hCG by RIA methods. Sinosich, M. et al reported an investigation of placental proteins in the diagnosis and evaluation of the "elusive" early pregnancy in Obstet.Gyn.Surv. 40:273-282 (1985). They found that hCG level determinations provide a false negative value of 17 percent, and SP1 value determinations were even more disappointing, 41 percent of proven ectopic pregnancies having no detectable SP1 level. Lower levels of PAPP-A with ectopic pregnancies was also reported.
HCG remains the most popular target for determining ectopic pregnancies. HCG is produced by fetal trophoblast and passes from the fetal blood into the mother's blood through the intervillous space in the placenta. HCG levels in maternal blood and urine are often detectable at about 3 weeks.
We have discovered that the status of normal uterine pregnancies can be determined early in the gestation cycle and with a high reliability by testing a sample for the presence of fetal restricted antigens, that is, antigenic or non-antigenic compounds or materials which are produced in the placental tissue and which do not pass in any substantial amounts into the maternal blood. Included in this class of antigens are fetal fibronectins. If the fetal restricted antigens are substantially depressed in a test sample from a pregnant patient, an ectopic pregnancy is indicated.